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2 Chapter 8 Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications of Neutrophil to Lymphocyte Ratio, Glasgow Prognostic Score and Related Parameters Ilze Strumfa, Tatjana Bogdanova, Arturs Kalva, Boriss Strumfs, Roberts Rumba, Andrejs Vanags, Inese Drike, Dzeina Mezale, Arnis Abolins, Arvids Jakovlevs, Dainis Balodis and Janis Gardovskis Additional information is available at the end of the chapter Abstract Gastric cancer induces systemic inflammatory reaction (SIR) manifesting with changes in counts of white blood cell fractions and concentrations of acute phase proteins, clotting factors and albumins. Thus, protein-based scores or blood cell ratios (neutrophil to lymphocyte ratio (NLR); platelet to lymphocyte ratio (PLR)) are used to evaluate SIR. SIR tests are biologically justified by multiple clinically important and fascinating events including bone marrow activation, development of immune-suppressing immature myeloid cells, generation of pre-metastatic niches and neutrophil extracellular trap formation from externalised DNA network in bidirectional association with platelet activation. Despite biological complexity, clinical SIR assessment is widely available, patient-friendly and economically feasible. Here we present concise review on NLR, PLR, Glasgow prognostic score and fibrinogen parameters that have prognostic role regarding overall, cancer-free and cancer-specific survival in early and advanced cases. Tumour burden can be predicted helping in preoperative detection of serosal or lymph node involvement. Practical consequences abound, including selection of surgical approach in respect to tumour burden, adjustments in treatment intensity by prognosis or evaluation of chemotherapy response. The chapter also scrutinises main controversies including different cut-off levels. Future developments should include elaboration of complex scores as described here. SIR parameters should be wisely incorporated in patients treatment. Keywords: gastric cancer, systemic inflammatory reaction, neutrophil to lymphocyte ratio, NLR, platelet to lymphocyte ratio, PLR, Glasgow prognostic score, fibrinogen 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

3 144 Gastric Cancer 1. Introduction Gastric cancer remains an important issue in world oncology. In 2013, it ranked fifth by the global incidence and second by mortality [1]. Although the death rates have decreased significantly in the USA and Europe over the previous 70 years, gastric cancer is characterised by poor prognosis and high mortality [2] except for early diagnosed cases. Thus, prognostic and predictive estimates are necessary to guide the intensity of treatment and to predict the effcacy of it. Different directions of prognostic evaluation have been studied, including classic means as tumour-node-metastasis (TNM) stage or patient s Eastern Cooperative Oncology Group (ECOG) performance status [3], or novel approaches as the molecular tests [4]. Many tumours, including gastric cancer, evoke systemic inflammatory reaction (SIR). The systemic effects of cancer include alterations in bone marrow function, especially myelopoiesis. The production and release of leukocytes increases. In addition, immature myeloid cells, including the precursors of granulocytes and monocytes, are retained in early stages of differentiation. Immature myeloid cells can act as immune suppressors and generate pre-metastatic niches, among other pathogenetic processes. Thus, it has even been stated that cancer is an inflammatory disease [5]. SIR shows complex associations with the local immune and inflammatory infiltrate in the tumour [6]. Cancer-related SIR involves cells of innate and adaptive immunity as well as soluble factors. Macrophages are recruited in tumour by hypoxia and tumour-released molecular agents including growth factors and cytokines [7]. Macrophage phenotype switch from tumoursuppressing classical M1 to tumour-promoting M2 subtype promotes angiogenesis and immunosuppression. Platelets undergo activation that contributes to cancer progression and patient mortality [8]. Neutrophil activation can stimulate angiogenesis and metastatic spread. Neutrophil extracellular traps formed from externalised DNA network are bidirectionally associated with platelet activation and can contribute to cancer progression via several mechanisms therefore neutrophil extracellular traps represent also an attractive treatment target [8]. Neutrophils are locally recruited in the cancer as well via chemokine signalling; they contribute to angiogenesis and increased blood vessel permeability. These molecular events highlight also the association between infection or surgery-induced inflammation [9, 10] and cancer relapse or metastatic spread. While innate immunity is generally thought to act as tumour enhancers, high numbers of infiltrating neutrophils [11] and macrophages [12] are shown to be protective in gastric cancer. In contrast, lymphocytes representing the adaptive immunity are considered to have tumour suppressing effects [7] although contrary effects have been ascribed to certain subpopulations [13, 14]. There is increasing body of evidence that patients survival can vary despite equal TNM parameters. In turn, cancer can cause systemic inflammatory response that might be associated with prognosis and/or response to treatment. SIR can be evaluated by number or ratio

4 Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications of serum neutrophils, lymphocytes, monocytes and platelets as well as by concentrations of acute phase proteins. These blood tests represent patient-friendly, widely available, globally standardised and cheap information that should be wisely incorporated in patients treatment [15]. Regarding the diagnostics of gastric cancer, several SIR parameters have been found to differ between gastric cancer patients and healthy controls. Such indicators include neutrophil to lymphocyte ratio [16 18], platelet to lymphocyte ratio, platelet count [18], mean platelet volume [18, 19] and red blood cell distribution width [18]. While these changes clearly indicate activation of systemic inflammatory reaction in gastric cancer, additional research is necessary to identify the diagnostic value of SIR parameters in the differential diagnosis between gastric cancer and other gastric pathologies, including precancerous, inflammatory and ulcerative changes. The correlation between neutrophil to lymphocyte ratio (NLR) and poor survival of gastric cancer patients is the best-known finding regarding SIR in gastric cancer [16, 17, 20]. High NLR is associated not only with shorter overall survival but also with worse progression-free survival [21]. In addition to the general association with survival, the prognostic value of NLR has been tested in specific clinical groups. Thus, NLR predicts post-operative survival of surgically treated patients with resectable cancer [22] and retains independent prognostic role in elderly patients an expanding group in Western population showing multiple ageing-related changes that could affect the immune and inflammatory processes [23]. For patients undergoing chemotherapy because of unresectable and recurrent advanced gastric cancer, NLR also shows independent prognostic significance [24]. NLR is an independent prognostic factor in metastatic gastric cancer [25] and in metastatic gastric cancer treated with chemotherapy [26]. The predictive value is limited in patients receiving palliative treatment for disseminated gastric cancer [21]. Some authors consider low NLR as an indicator for good prognosis and thus beneficial effect of surgical treatment in stage IV gastric cancer [27, 28]. Some research groups have found that complex assessment of SIR-related parameters has superior prognostic value. For instance, joint analysis of platelet count and NLR was found to predict post-operative survival more exactly [29]. Combined scoring of albumin and neutrophil to lymphocyte ratio was independently associated with overall survival and was especially accurate for patients with stage I II gastric cancer [30]. Combined evaluation of pre-operative NLR and platelet to lymphocyte ratio (PLR) was independent predictor of survival after curative surgical resection of stage I II gastric cancer [31]. SIR can highlight wider scope of clinical traits, including manifestations that are not directly related to surgery or oncologic treatment. For example, pre-operative anxiety and depression are significantly associated with NLR [32]. SIR assessment is more comprehensive than NLR analysis. Thus, pre-operative plasma fibrinogen increases with gastric cancer stage and predicts worse recurrence-free and overall survival [33]. Similarly, levels of plasma albumin or the characteristics of platelets can provide significant data. Levels of C-reactive protein, original or modified Glasgow prognostic score can be used for analysis [3, 15].

5 146 Gastric Cancer The systemic inflammatory reaction itself can be an adverse pathogenetic event, facilitating tumour angiogenesis or adhesion of circulating tumour cells to endothelium that would lead to the growth of metastasis. In addition, NLR correlates with other factors known to have adverse prognostic role. Among such parameters, presence of vascular and lymphatic invasion as well as positive resection lines have been reported [22]. In several studies, NLR has been found to correlate with the stage of gastric cancer [16, 20 22]. NLR negatively correlates with mismatch repair protein deficiency [34]. NLR is associated with post-operative infectious complications. Both factors show an independent significant association with poorer survival after gastrectomy [9]. The evaluation of SIR in gastric cancer patients is highly attractive. By increasing awareness of SIR parameters, simple and widely available blood tests can provide information that is helpful in shaping the care of gastric cancer patients from early stages to metastatic spread or locally advanced tumour. However, unresolved issues remain. Except the prognostic value to NLR, many aspects as the correlation with tumour morphology, type by Lauren classification, invasive properties of cancer, grade, intensity of angiogenesis and microvascular density have been targeted by low number of studies. Only few meta-analyses have been conducted [21, 35 37]. Few data are available on SIR parameters after treatment although it is known that post-chemotherapy NLR correlates with the response in patients with unresectable gastric cancer [38]. The practical unsolved questions include the comparison between NLR and other indicators of systemic inflammatory response, e.g., platelet to lymphocyte ratio [39], the significance of post-treatment NLR as well as cut-off values for practical use. The ultimate goal would be to create and validate an algorithm for fine-tuning of the treatment strategy in gastric cancer from early to advanced stages. Inflammatory markers other than NLR should be included; complex assessment hypothetically could be advised. Thus, considering the high incidence and mortality of gastric cancer and the need for prognostic and predictive data, the present chapter will be devoted to the assessment of SIR in gastric cancer in order to develop practical recommendations how to adjust gastric cancer treatment by easily available and economically feasible simple blood tests for SIR parameters. Increased awareness of SIR characteristics is important to reach this aim. 2. Neutrophil to lymphocyte ratio in gastric cancer Neutrophil to lymphocyte ratio is calculated as the ratio between the count of neutrophilic leukocytes and lymphocytes in peripheral blood. Thus, the parameter is easily available, especially in carefully examined cancer patients, and economically non-demanding. In fact, suffcient awareness and algorithm for interpretation are the only prerequisites to obtain an additional piece of information from routine blood tests. Since the early reports [40, 41], NLR has been studied in relation to the prognosis of gastric cancer patients. Thus, Aliustaoglu et al. reported that high NLR was statistically

6 Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications significantly associated with shorter median survival. In the same study, similar association was found regarding high platelet to lymphocyte ratio and high absolute number of lymphocytes but no difference was found for neutrophil count, platelet count and mean platelet volume [41]. In another early study devoted to the prognostic significance of hostand tumour-related factors in patients with gastric cancer, white blood cell count, NLR, C-reactive protein (CRP) and albumin was found to have prognostic impact, along with age, haemoglobin level, tumour size as well as T and N characteristics. By multivariate analysis, NLR was an independent prognostic factor along with tumour size and T parameter [42]. At present, the association between NLR and different aspects of survival (overall, cancerspecific, cancer-free or progression-free survival) remains one of the best substantiated aspects in the SIR research in gastric cancer NLR and survival: prognostic implications The prognostic importance of NLR is shown over the whole course of gastric cancer, and is applicable to wide treatment spectrum from surgically resectable cases, including early gastric cancer, to advanced, recurrent or metastatic tumours subjected to non-surgical treatment. Most researchers have demonstrated that NLR is an independent prognostic factor, based on multivariate analysis [17]. However, in few studies, the association with survival is confirmed by univariate but not multivariate analysis [43 45]. Some of the reports are on better scores, e.g. Glasgow prognostic score had higher informativity in a large and homogeneous group of 324 patients with stage III gastric cancer undergoing resection [43]. The prognostic value of NLR has been reported in different cancers, including lung, colorectal and breast carcinoma, among others [46]. Gastric cancer also follows the same mechanisms. In unselected cohort of patients diagnosed with gastric adenocarcinoma, high NLR (compared with the cut-off value 3) was a significant (p = 0.016), independent risk factor for poor survival [17]. Surgery is the mainstay of gastric cancer treatment, if the local and/or systemic tumour spread, or the general condition of the patient does not limit the possibilities of surgical intervention. In patients who have had curative surgery for gastric cancer, high NLR is significantly associated with poor prognosis [39], including overall survival [16, 47 49], cancer-specific survival [47], cancer-free survival [16, 47] and progression-free survival [25, 38, 50]. Thus, in a recent study of 162 patients with resectable gastric cancer, high pre-operative NLR (reaching or exceeding the median of 4.02) was associated with decreased overall and cancer-free survival, confirmed by Kaplan-Meier analysis [16]. In a significantly larger group of 1986 consecutive patients subjected to curative surgical treatment for gastric cancer, NLR was confirmed as an independent prognostic factor for overall survival, associated with hazard ratio of 1.4 [39]. Similarly, in 601 surgically treated gastric cancer patients, high NLR (reaching or exceeding 1.7) was a significant prognostic parameter for overall survival, confirmed as an independent factor by multivariate analysis. The hazard ratio was 2.12 [48]. Analogous observations were reported by Hsu et al. They assessed a large cohort of 1030 gastric cancer patients subjected to complex treatment. In accordance with clinical indications,

7 148 Gastric Cancer subtotal or total gastrectomy along with spleen- and pancreas-sparing D2 lymphadenectomy was performed, aiming to accomplish clear resection margins. Metastasectomy was considered depending on clinical symptoms and possibility of radical resections, and adjuvant or palliative chemotherapy was offered for stage II IV patients. In such a large group, showing the routine clinical diversity of gastric cancer presentation, high NLR (exceeding 3.44) was an independent prognostic factor for overall survival, associated with hazard ratio of 1.57 [22]. In addition to significant statistical findings, the biological differences between groups also are remarkable. The 3- and 5-year survival rates in low versus (vs.) high NLR groups were 71.0% vs. 55.1% and 64.1% vs. 47.2%, respectively [22]. Even more, the 5-year survival was 29.9% in the high NLR group (reaching or exceeding 5.0) contrasting with statistically significantly different value of 85.6% in patients who had low NLR [51]. The overall survival was 86.1 months in patients presenting with low NLR vs months in high NLR (reaching or exceeding 2.3) group [30]. Evaluating 156 surgically treated gastric cancer patients, the median survival in high vs. low NLR groups was 36 vs. 60 months while the five-year survival was 35% and 60%, and the median cancer-free survival was 12 and 20 months, respectively. The survival differences retained significance in N0 patients: 5-year survival was 60% vs. 90%, p < In this cohort, NLR was also recognised as an independent prognostic factor for overall survival [52]. In advanced gastric cancer (stage III IV) patients subjected to gastrectomy with curative intent, high NLR was an independent predictor of overall survival at cut-off 2.0 corresponding to median while cut-off value 3.0 (the 75th percentile) was an independent predictor of cancer-free survival. The median overall survival in high vs. low NLR was 21.4 and 45.3 months while the progression-free survival in the redefined high and low NLR groups was 12.8 vs months [53]. NLR retains prognostic significance for surgically treated gastric cancer patients in specific subgroups. For instance, in elderly gastric cancer patients (aged 75 years or older) treated by gastrectomy, high NLR (reaching or exceeding 1.83) was associated with worse survival. Again, NLR was confirmed as an independent risk factor by multivariate analysis. The biological differences were remarkable: the median survival associated with low vs. high NLR was 1209 vs. 587 days, respectively [16]. High NLR is associated with older age in some studies [9, 20, 44, 47, 54, 55] while others report no association [22, 38]. It is very important to identify high risk of cancer progression in early diagnosed cases. Some promising reports have been published. Combined score including NLR and albumin level was shown to have independent prognostic value exceeding the informativity of NLR as justified by higher area under curve (AUC). This score, further described in detail, retained the prognostic ability in stage I II gastric cancer [30]. A complex score comprising NLR and PLR is another prognostic option, successfully tested in a stage I II gastric cancer. NLR-PLR score showed a clear trend to improve the prognostic value of TNM staging [31]. Mohri et al. has reported very interesting findings regarding NLR in surgically treatable gastric cancer cases. In 404 patients undergoing curative gastrectomy for gastric cancer, high NLR

8 Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications was an independent risk factor of post-operative infectious complications while it was not predictive of non-infectious complications. In turn, both high NLR and post-operative infectious complications were independent risk factors of worse overall and cancer-specific survival [9]. The preceding NLR increase in patients later developing post-operative infectious complications but not in case of all complications was justified by Japanese scientists [10]. In contrast with the previously described findings, NLR was not informative regarding survival of gastric cancer patients having only local disease while it was significantly associated with survival in advanced cases [56]. Some negative findings, including the cited one, can be explained by small study group comprising only 53 patients with local disease and 50 with advanced cancer [56]. Evaluating Glasgow prognostic score, NLR and PLR in patients with resected stage III gastric adenocarcinoma, only Glasgow prognostic score along with TNM stage was independently associated with cancer-free and overall survival [43]. If the study design includes several SIR parameters, multivariate analysis could highlight only one of those. Advanced or metastatic cancer represents a situation with continuously significant tumour burden, associated with ongoing inflammation, angiogenesis, antigenic stimulation and thus sustained SIR. The NLR has been evaluated in these situations as well. In 174 advanced gastric cancer patients treated with oxaliplatin/5-fluorouracil (FOLFOX), NLR was associated with overall survival but not with progression-free survival. NLR was also an independent predictor of overall survival. Normalisation of NLR after one cycle of chemotherapy was significant and independent predictor of overall and progression-free survival [57]. Similar findings are reported by Jin et al. [58]. In unresectable and recurrent advanced gastric cancer patients treated by chemotherapy, high NLR (exceeding 4) was associated with significantly lower median survival [24]. Similarly, in another cohort comprising 143 cases of metastatic gastric cancer, high NLR was an independent prognostic factor. The overall and progression-free survival was 11.6 and 7.9 months in low NLR (less than 3.34) group contrasting with 8.3 and 6.2 months in patients having high NLR [25]. In 120 unresectable metastatic and advanced gastric cancer cases, treated by chemoradiotherapy, baseline NLR predicted survival. The median overall and progression-free survival in high vs. low NLR group was 10 and 3 months vs. 18 and 6 months. Treatment-induced changes in NLR also predicted survival. Both baseline NLR and changes upon initiation of treatment predicted treatment outcomes [38]. This finding is in accordance with Cho et al., who also reported significantly higher chemotherapeutic disease control rate in metastatic advanced gastric cancer patients having low NLR, defined as less or equal to 3.0 [50]. Combined scores have been generated to evaluate the prognosis of metastatic gastric cancer as well [26]. Occasionally NLR shows association with survival by univariate but not multivariate analysis. Thus, in a small group of 70 patients affected by locally advanced gastric cancer (stage III IV) and treated by neoadjuvant chemotherapy, NLR was an independent predictor of overall survival. It was significantly associated with progression-free survival but was not an independent factor [59]. In a large group of 439 patients affected by metastatic or recurrent gastric cancer, NLR was significantly associated with overall survival in univariate but not multivariate analysis. Complex score was favoured by authors [60]. The prognostic findings regarding NLR in gastric cancer have been summarised in Table 1.

9 150 Study group Survival References Characteristics Size Overall Cancer-specific Cancer-free Progression-free Unselected gastric adenocarcinoma 706 X [17] Gastric Cancer GC 245 X Kaplan-Meier analysis of multicentre study data Gastric adenocarcinoma 236 X [61] [62] Surgically treated GC Consecutive GC patients undergoing curative gastrectomy 404 X X [9] Curative surgery for GC 288 X Resectable GC 162 X Kaplan-Meier analysis X Kaplan-Meier analysis [49] [16] GC, subjected to curative surgery 1986 X [39] Surgically treated GC (R0) 601 X [48] GC patients undergoing gastrectomy 389 X X X [47] Surgically treated GC patients 207 X X [63] GC subjected to radical surgery 291 X [20]

10 Study group Survival References Characteristics Size Overall Cancer-specific Cancer-free Progression-free GC subjected to gastrectomy GC subjected to potentially curative gastrectomy 632 X Univariate (significant) (NS) 156 X [45] [52] Patients with resectable GC, including advanced cases Surgically treated (total or subtotal gastrectomy) GC patients Gastrectomy with curative intent for stage III IV GC 377 X 220 X Univariate (significant) (NS) 293 X Curative gastrectomy 157 X GC patients, undergoing gastrectomy Elderly patients (at least 75 years old) undergoing gastrectomy Curative resection, D2 lymphadenectomy, adjuvant chemotherapy in stage II III Resectable GC subjected to combined treatment 1028 X 160 X 873 X Kaplan-Meier analysis 1030 X X [64] [44] [53] [65] [66] [23] [30] [22] Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications

11 152 Study group Survival References Characteristics Size Overall Cancer-specific Cancer-free Progression-free Advanced, unresectable and/or metastatic GC Gastric Cancer Unresectable and recurrent advanced GC, treated by chemotherapy Metastatic GC treated by chemotherapy 224 X 256 X [24] [26] Metastatic GC 143 X X [25] Unresectable, advanced GC, treated by chemotherapy 120 X Kaplan-Meier analysis X Kaplan-Meier analysis [38] Metastatic GC treated by chemotherapy 109 X Kaplan-Meier analysis X Kaplan-Meier analysis [67] GC, stage IV with synchronous distant MTS 123 X [27] Metastatic advanced GC treated by palliative chemotherapy 268 X X [50] Locally advanced GC treated by neoadjuvant chemotherapy 70 X X Univariate (significant) (NS) [59] Metastatic or recurrent GC 439 X Univariate (significant) (NS) [60]

12 Study group Survival References Characteristics Size Overall Cancer-specific Cancer-free Progression-free Inoperable advanced or metastatic GC patients receiving chemotherapy 384 X Univariate (significant) (NS) [68] Advanced GC patients treated by chemotherapy 174 X X (dynamics, not baseline) [57] Advanced GC treated with neoadjuvant chemotherapy Metastatic unresectable advanced GC patients treated with palliative chemotherapy 46 X (baseline and dynamics) X Univariate (significant) (NS) 104 X Abbreviations: GC, gastric cancer; R0, resection line free of cancer; NS, not significant; MTS, metastasis. Table 1. The prognostic value of NLR in gastric cancer patients. X (baseline and dynamics) [58] [69] Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications

13 154 Gastric Cancer The cut-off levels vary widely among the studies. Most frequently, either the median value is selected as the cut-off [16, 70], or the relevant level is found by receiver operating characteristic curve (ROC) analysis [30, 39]. Youden Index has been successfully employed to detect the optimal cut-off during ROC analysis [30]. This index is defined as the cut-off value showing the highest sum of specifity and sensitivity at the considered value; minus 1 [71]. Less frequently, the 75th percentile is used as the cut-off [44, 53]. Some research groups have applied more complex approach, e.g. combining the patients groups with similar survival [17, 20]. The reported cut-off levels for NLR in gastric cancer patients are summarized in Table 2. Interestingly, different cut-off values can reveal different information. Thus, Jung et al. has reported that cut-off 2.0 based on the median value was valuable in order to show that higher NLR is an independent risk factor for worse overall survival. However, when studying cancer-free survival, NLR was an independent risk factor by cut-off 3.0 corresponding to the 75th percentile [53]. The necessity for different cut-offs in regard to the question of interest is indirectly demonstrated by mean NLR in different patient groups: 4.02 in T1 2; 6.54 in T3 4; 4.81 in N0; 6.41 in N+; 5.00 in M0; 7.82 in M1; 4.74 in stage I II cancers and 7.07 in stage III IV cancers [47]. Jung et al. also observed statistically significant differences in median NLR by gastric cancer stage: 1.88 in stage III and 2.17 in stage IV [53] Association with tumour features Local tumour spread: T Significant association between NLR and the invasion depth of gastric cancer is recognised since the early studies [65] and confirmed by more recent research [20]. The applied cut-off levels again vary widely. Thus, the association with increased depth of invasion has been demonstrated in patients whose high preoperative NLR level was defined as higher than or equal to 4.02 [16] or as exceeding the ROC-set cut-off value of 1.59 [55]. Significant difference in T1 2 vs. T3 4 distribution was reported by Deng et al. The mean NLR was 4.02 in T1 2 cases and 6.54 in T3 4 cases [47]. Many studies have highlighted the association between NLR and serosal invasion that is classified as T4a. Such invasion represents a potential limit to surgical treatment if followed by extensive peritoneal spread. NLR studies in regard to the tumour spread have led to the development of complex predictive scores to forecast serosal invasion. Hence, high NLR can be used as an independent predictive factor for T4 using cut-off 3.2 [73]. The high NLR (exceeding 3.44) group had significantly higher proportion of T4 when 1030 patients with resectable gastric cancer were assessed [22]. Serosal invasion was significantly more frequent in elderly patients having high NLR: 75.5% vs. 57.4% [23]. Finally, in a large prospective study enrolling 1131 surgically treated patients, high NLR (exceeding the median 3.5) was associated with deeper invasion: T3 T4 tumours. The mean NLR was 2.51 in T3 T4 tumours vs in T1 T2 tumours. Within the frames of a complex score, NLR can be used to predict inappropriateness of gastrectomy [54]. The capacity of NLR to predict such tumour spread that would limit surgical treatment has been explored in combined model searching for either peritoneal or metastatic spread due to either gastric or oesophageal adenocarcinoma. Authors concluded that NLR reaching or exceeding

14 Study group Cut-off Study target Level of justification Characteristics Size Value Approach Main conclusions References Unselected gastric adenocarcinoma Complex OS Higher NLR is associated with worse OS [17] GC Ref. [40] OS Kaplan-Meier analysis Multicentre study Surgically treated GC High NLR is significantly associated with worse OS, presence of N+ and higher stage Operable GC ROC analysis N High NLR shows significant association with N+ in early GC but is not an independent factor Stage I II GC, subjected to radical (R0) surgery including D2 lymphadenectomy Stage I II GC, subjected to radical (R0) surgery including D2 lymphadenectomy Surgically treated T2 GC ROC analysis OS Within the frames of complex NLR- PLR score is an independent predictor of OS in stage I II GC Ref. [29] OS Within the frames of complex score, including platelet count and NLR, is not the most informative predictor of OS by AUC assessment Median N Higher NLR is associated with higher number of LN MTS and higher N [61] [72] [31] [31] [70] Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications

15 156 Study group Cut-off Study target Level of justification Characteristics Size Value Approach Consecutive patients undergoing curative gastrectomy ROC analysis OS CSS Post-operative complications Main conclusions Higher NLR is an independent risk factor for worse OS, CSS and postoperative infectious complications References [9] Gastric Cancer Curative surgery for gastric cancer ROC analysis for survival OS Immune cell density within cancer Higher NLR is an independent risk factor for worse OS Density of CD4 Ly is decreased in high NLR group while CD3 and CD8 + Ly density shows no differences [49] Operable GC ROC analysis N High NLR is an independent factor, associated with N+ [55] Curative resection, D2 lymphadenectomy, adjuvant chemotherapy in high-risk stage II III ROC analysis OS Kaplan-Meier analysis Although NLR is associated with OS, a complex score including NLR and albumin is more potent predictor of OS based on higher AUC in ROC analysis [30] Elderly patients (at least 75 years old) undergoing gastrectomy ROC analysis OS Higher NLR is an independent risk factor for worse OS [23] Surgically treated GC ROC analysis OS Higher NLR is an independent risk factor for worse OS [48]

16 Study group Cut-off Study target Level of justification Characteristics Size Value Approach Main conclusions References Total or subtotal gastrectomy with lymphadenectomy ROC analysis OS, CFS, CSS Higher NLR is a significant risk factor for worse OS, CFS, CSS [47] Resectable GC Median OS, CFS Kaplan-Meier analysis Higher NLR is an associated with worse OS and CFS [16] Resectable gastric cancer subjected to combined treatment Survival tree assessment by R software Surgically treated GC 207 5/4 ROC analysis OS CFS GC subjected to radical surgery GC subjected to gastrectomy GC subjected to potentially curative gastrectomy Complex assessment of survival by NLR intervals 3 and 5-year OS rate OS Tumour and patients characteristics Higher NLR is an independent risk factor for worse OS Higher NLR is an independent risk factor for worse OS. However, GPS has higher prognostic value High NLR is an independent prognostic factor for overall survival and is significantly associated with, age, tumour size, T and TNM stage ROC analysis OS High NLR shows significant association with OS but is not an independent factor. Complex score preferred Median OS Higher NLR is an independent risk factor for worse OS [22] [63] [20] [45] [52] Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications

17 158 Study group Cut-off Study target Level of justification Characteristics Size Value Approach Surgically treated GC, including non-radical cases Median Resection line status Tumour characteristics Mann Whitney test Fisher test Univariate analysis Main conclusions High NLR is associated with T3-4, G3-4, larger tumours, higher N and TNM stage Within frames of complex score NLR can be used to predict inappropriateness of gastrectomy References [54] Gastric Cancer Surgically treated GC patients Surgically treated GC, T2 4 Gastrectomy with curative intent for stage III IV GC Gastrectomy with curative intent for stage III IV GC th percentile OS Higher NLR is a significant risk factor for OS by univariate but not multivariate analysis ROC analysis T4 High NLR is an independent factor, associated with T Median OS Higher NLR is an independent risk factor for worse OS th percentile CFS Higher NLR is an independent risk factor for worse CFS [44] [73] [53] [53] Curative gastrectomy Refs. [74, 75] CSS Higher NLR is an independent risk factor for worse CSS [65] Advanced, unresectable and/or metastatic GC Metastatic gastric adenocarcinoma treated by chemotherapy Refs. [53, 66] OS NLR is an independent risk factor [26]

18 Study group Cut-off Study target Level of justification Characteristics Size Value Approach Main conclusions References Metastatic GC Median OS Higher NLR is an independent risk factor for worse OS [25] Metastatic GC treated by chemotherapy Refs. [40, 58] OS PFS Kaplan-Meier analysis High NLR is significantly associated with worse OS and PFS [67] Unresectable, advanced GC, treated by chemotherapy Unresectable, advanced GC, treated by chemotherapy Advanced GC treated by chemotherapy// Local GC treated by surgery and adjuvant chemoradiotherapy GC (stage IV) with synchronous distant MTS Median OS PFS Median Response to chemotherapy Kaplan-Meier analysis χ 2 test 50// Median OS Kaplan-Meier analysis Higher baseline NLR or increase of NLR after first-line chemotherapy is associated with worse OS and CFS Lower baseline NLR or lower NLR after firstline chemotherapy was associated with improved response to chemotherapy High NLR is significantly associated with worse OS in advanced but not local GC Median OS Higher NLR is a significant risk factor for worse OS in the whole group and in surgically treated patients [38] [38] [56] [27] Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications

19 160 Study group Cut-off Study target Level of justification Characteristics Size Value Approach Metastatic advanced GC treated by palliative chemotherapy Median OS, PFS Response to chemotherapy Main conclusions Higher NLR is an independent risk factor for worse response to chemotherapy, OS and PFS References [50] Gastric Cancer Inoperable advanced and metastatic GC patients receiving palliative chemotherapy Median OS High NLR shows significant association with OS but is not an independent factor [68] Advanced GC patients treated with chemotherapy OS curve analysis OS PFS Low baseline NLR and normalisation of NLR were independent predictors of better OS. Normalisation of NLR was an independent predictor of better PFS. [57] Advanced GC treated by neoadjuvant chemotherapy Ref. [40] OS PFS [58] Abbreviations: OS, overall survival; NLR, neutrophil to lymphocyte ratio; GC, gastric cancer; Ref., reference; N+, presence of metastases in regional lymph nodes; ROC, receiver operating characteristic curve; N, regional lymph node status in respect to metastases by tumour-nodes-metastasis (TNM) classification; R0, resection line free of tumour; PLR, platelet to lymphocyte ratio; AUC, area under the curve; T, local spread of primary gastric cancer by TNM classification; LN, lymph node; MTS, metastasis; CSS, cancer-specific survival; CD, cluster of differentiation; Ly, lymphocyte; CFS, cancer-free survival; GPS, Glasgow prognostic score; TNM, tumour-nodes-metastasis classification; G, grade; PFS, progression-free survival. Table 2. Cut-offs of NLR in gastric cancer studies.

20 Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications the cut-off value of 3.28 is an independent predictor of undesirable tumour spread. The median NLR in operable patients vs. those having peritoneal or metastatic disease was 2.2 vs. 3.3 [76]. Negative findings have been published. Some of them could be easily explained by small group size, e.g. only 61 gastric cancer patients were enrolled in the study of Pietrzyk et al. [18]. However, no differences in T distribution by NLR were found by Kim et al. who analysed a large group of 601 patients [48]. No association between invasion depth and NLR was found in a multicentre study [61]. Large tumour size has shown association with high NLR [20, 22, 38, 53 55, 65, 77]. As T in gastric cancer is not defined by size, tumour size could become a confounding factor Metastases in regional lymph nodes: N Metastatic involvement of regional lymph nodes is associated with worse prognosis, being especially important in the early stages of gastric cancer. Presence of lymph node metastases also limits and changes the treatment options as endoscopic resection is not feasible anymore but D2 lymphadenectomy becomes more appropriate than D1 lymphadenectomy. In addition, neoadjuvant treatment can be offered now to gastric cancer patients affected by lymph node metastases [55]. NLR can be used to predict lymph node metastasis. In a retrospective study of 230 surgically treated patients, affected by T2 gastric cancer, NLR exceeding the median value of 2.18 was associated with higher number of lymph node metastases and higher N characteristics. The findings were confirmed by multivariate analysis. The relative risk was as high as 4.15 and 7.09 in regard to high number of metastases and N stage, respectively [70]. NLR at the cut-off level 1.59 (detected by ROC) was an independent factor associated with lymph node metastasis; however, higher informativity reflected by higher AUC was achieved by complex score (see further) including NLR, PLR and tumour-related factors [55]. The conclusions are justified by other researchers reporting correlation between NLR and N parameter since the early reports [65] until recent studies [77]. Thus, high NLR (exceeding the ROC-set cut-off value of 1.59) was associated with high N [55] while low preoperative NLR level (less than 4.02) was associated with lower number of lymph node metastases [16]. The variability of applied cut-off values is evident. Lymph node metastases were significantly more frequent in elderly patients having high NLR: 83.0% vs. 55.6% [23]. In a large cohort of 1030 patients with resectable gastric cancer, high ratio of metastatic to examined lymph nodes defined as exceeding 0.18 was more frequent in those who had high NLR (greater than 3.44). Interestingly, in the same study N distribution showed only a trend to differences [22]. Significant difference in N0 vs. N+ distribution was reported by Deng et al. In addition, the mean NLR was 4.81 in N0 patients and 6.41 in N+ cases [47]. Statistically significant correlation between presence of lymph node metastasis, high NLR was confirmed in a multicentre study [61]. In a prospective study of 1131 surgically treated cases, high NLR (exceeding the median 3.5) was associated with higher N. The mean NLR was 2.31 in N0; 2.32 in N1; 2.43 in N2 and 2.75 in N3 cases [54]. Negative findings have been published as well. Some of them could be easily explained by small group size, e.g. only 61 gastric cancer patients were enrolled in the study of Pietrzyk

21 162 Gastric Cancer et al. [18]. No differences in N distribution by NLR were found by Kim et al. who analysed a large group of 601 patients [48] and Yu et al. who assessed another significant cohort of 291 patients. In the same study, association with T and TNM stage was significant [20]. There was no correlation between NLR and N in a reasonable group of 262 surgically treated patients affected by T2 T4 gastric cancer while correlation with T in the same study was meaningful. The cut-off in this study was detected by ROC and was 3.2 [73]. Some reports have re-evaluated the meaning of NLR in predicting N status, arriving to less positive conclusions. In early gastric cancer (T1a T1b), NLR was significantly associated with presence of lymph node metastases. The mean NLR was 2.07 in N0 group while it increased to 2.60 in N+ group. However, by multivariate analysis NLR was not an independent prognostic factor. Complex score not including NLR was more informative for preoperative estimation of lymph node metastases [72] Presence of distant metastases: M Presence of distant metastasis has also been associated with higher NLR [38, 77]. Metastatic tumours were significantly more frequent in patients who had high NLR (exceeding 3.44) assessing 1030 patients with resectable gastric cancer [22]. Significant difference in M0 vs. M1 frequencies by NLR groups was reported by Deng et al. In addition, the mean NLR was 5.00 in M0 cases and 7.82 in M1 cases [47]. In a large study of 491 gastric cancer patients, NLR was significantly associated with peritoneal metastasis. However, it was not an independent predictive factor for peritoneal spread, while tumour morphology, serum level of carbohydrate antigen CA19-9 and lymphocyte count retained independent predictive value [78]. In contrast, evaluating CRP, activated partial thromboplastin time, NLR and hypoalbuminemia, NLR was identified as an independent risk factor of the presence of peritoneal metastasis. The cut-off level was set at 2.37 [79] TNM stage Considering the previously discussed links between NLR and TNM parameters, correlation with TNM stage could be expected as well. Indeed, advanced TNM stage was significantly associated with high NLR [9, 20, 44, 47, 65, 77]. High NLR (exceeding the ROC-set cut-off value of 1.59) was associated with high TNM stage [55]. The mean NLR was 4.73 in stage I II and 7.07 in stage III IV [47]. In advanced gastric cancer (stage III IV) patients, there still was difference between stage III and IV [53]. Statistically significant correlation between cancer stage and high NLR was confirmed also by multicentre [61] and prospective study design [54]. In a prospective study of 1131 surgically treated patients, high NLR (exceeding the median 3.5) was associated with higher TNM stage. The mean NLR was 2.13 in stage I, 2.40 in stage II, 2.53 in stage III and 2.60 in stage IV [54]. Regarding negative reports, no NLR differences by TNM stage were found by Kim et al. who analysed a large group of 601 patients [48].

22 Systemic Inflammatory Reaction in Gastric Cancer: Biology and Practical Implications Histological type and grade (G) The association between NLR and cancer grade is more controversial. The cancer grade was not different between high and low NLR groups in a cohort of 143 metastatic gastric cancer cases as well as in 389 patients who underwent gastrectomy or in 293 gastric cancer patients diagnosed in stage III IV [22, 25, 47, 53]. No difference by differentiation degree (G1 2 vs. G3) was found by Yu et al. [20]. In contrast, high NLR was associated with differentiated (vs. undifferentiated) gastric cancer [9]. High differentiation degree (vs. moderate and poorly differentiated cases) was associated with low NLR. In the same study, no differences were observed regarding proliferation fraction by Ki-67 [38]. High NLR (exceeding the ROC-set cut-off value of 1.59) was associated with high grade [55]. In a prospective study of 1131 surgically treated patients, high NLR (exceeding the median 3.5) was associated with poor differentiation or undifferentiated tumours while low NLR with high and moderate differentiation. The relevant mean NLR values were 2.46 in G3 G4 vs in G1 G2 cancers [54]. There was no correlation between NLR and histological differentiation in a large group of 262 surgically treated patients affected by T2 T4 gastric cancer while correlation with T in the same study was meaningful. The cut-off in this study was detected by ROC and was 3.2 [73]. No correlation between histological type of cancer and NLR was observed in a prospective study of 1131 surgically treated patients [54]. No differences in histology distribution by NLR were found by Kim et al. who analysed a large group of 601 patients [48]. Histological types (papillary, tubular, poorly differentiated, mucinous, signet ring cell carcinoma) were scrutinized by Deng et al., also finding no association with NLR level [47]. No NLR differences were observed between Lauren types: intestinal vs. diffuse [38, 53, 65] that might explain the lack of association with HER-2 protein expression [38]. Low NLR shows significant correlations with mismatch repair deficiency [34]. In cancer tissues, the density of CD4-positive lymphocytes was significantly decreased in high NLR group while the density of CD3 and CD8-positive lymphocytes was not associated with NLR [49]. Although NLR correlated with survival, it did not correlate with tumourinfiltrating lymphocytes [62]. Regarding cytokines and angiogenic factors, serum levels of osteopontin and interleukin 6 were significantly associated with NLR in gastric cancer patients [80]. NLR is significantly associated with helper T lymphocyte Th1/Th2 ratio in blood [65] Manifestations of invasive growth Only few scientists have assessed the relations between NLR and such manifestations of invasive growth as perineural, lymphatic and vascular invasion. Theoretically, such association could be hypothesised on the basis of prognostic value of NLR and the correlations between NLR and metastatic cancer spread. However, at present, negative reports predominate although are not unequivocal.

23 164 Gastric Cancer The frequency of perineural growth was not different between high and low NLR groups [22]. The frequency of lymphovascular invasion also was not different between high and low NLR groups in a cohort of 143 metastatic gastric cancer cases [25]. In contrast, vascular or lymphatic invasion was significantly more frequent in patients who had high NLR (exceeding 3.44) assessing 1030 cases of resectable gastric cancer. Hypothetically, the higher capacity for invasive growth could be the reason of more frequent occurrence of R1 in patients presenting with high NLR. However, association between NLR and resection line status (R0 vs. R1 vs. R2) was found by Jung et al., who observed no differences in the frequency of lymphatic, vascular and perineural growth regarding NLR level [53] The diagnostic role of NLR and confounding factors Several haematological parameters, including NLR, are significantly higher in gastric cancer patients than in healthy individuals [18]. A number of studies have confirmed that patients affected by gastric carcinoma have significantly higher NLR than healthy controls [16, 17]. NLR was also higher in gastric cancer patients if compared with persons having adenoma or benign gastrointestinal stromal tumour: 2.17 vs Excluding the confounding factors, NLR was an independent predictor of gastric cancer, associated with the odds ratio of 1.446, p = [77]. NLR is influenced by smoking [81]. Such differences are reported in gastric cancer patients as well [25] while other researchers have found no difference [47]. Non-oncological diseases, including both inflammations and such frequent non-inflammatory pathologies as diabetes mellitus and atrial fibrillation, among others, can also influence NLR [82]. Thus, SIR should be assessed within the frames of complex patient evaluation Meta-analyses of NLR in gastric cancer Several meta-analyses of NLR in gastric cancer have been carried out. Sun et al. have assessed 19 studies of NLR in gastric cancer. They confirmed the association between high NLR and worse overall, progression- or cancer-free survival, and higher stage. The predictive role was lost for stage IV patients who received palliative surgery only [21]. Nineteen studies were subjected to meta-analysis by Xin-Ji et al. [37]. Elevated NLR was associated with shorter overall (odds ratio (OR) 1.65; 95% CI = ) and shorter cancer-free survival (OR 1.61; 95% CI = ). Regarding the tumour characteristics, NLR was associated with presence of lymph node metastasis, and high T (T3 + T4) and high stage (III IV). The odds ratio for lymph node metastasis, 1.70 (95% CI = ), for T3 or T4 cancer 2.93 (95% CI = ) and for stage III IV: 1.87 (95% CI = ) as reported by Xin-Ji et al. [37]. By meta-analysis performed by Chen et al. [36], high NLR was associated with poor overall survival (hazard ratio (HR) 2.16; 95% CI = ) and progression-free survival (HR 2.78; 95% CI = ). In a meta-analysis of 10 studies, higher NLR was associated with worse overall (HR 1.83; 95% CI = ), progression-free (HR 1.54; 95% CI = ) and cancerfree (HR 1.58; 95% CI = ) survival [35].